RU2703805C1 - Method for human genotype determination by polymorphism ugt1a1*6/*28 in promoter region of ugt1a1 gene - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, specifically to molecular biology and genetics, and can be used to determine a human genotype by UGT1A1*6/*28 (7TA/6TA) polymorphism in the promoter region of the UGT1A1 gene. Method involves application of specific oligonucleotide sequences with registration of results of CPR in real time using the method of analysis of melting curves (HRM). Reaction mixtures include specific primers, as well as an adapted buffer for Taq-polymerase, which increases sensitivity of the method.

EFFECT: invention makes it possible to reduce costs for molecular-genetic research, to increase its availability and accuracy of testing due to use of original oligonucleotide sequences and optimized reaction mixtures.

1 cl, 1 dwg

Description

The proposed method relates to the field of biotechnology, molecular biology and genetics, and can be used to determine the human genotype by polymorphism UGT1A1 * 6 / * 28 in the promoter region of the UGT1A1 gene with the test sample assigned to a heterozygous or homozygous diagnostic allelic variant (genotypes 6TA / 6TA , 6TA / 7TA7, 7TA / 7TA).

Pharmacogenetic testing to predict the development of toxic and side effects during drug treatment is one of the rapidly developing areas in DNA testing and is gradually taking its place in many clinical guidelines for the treatment of malignant neoplasms. The development and implementation of a method for testing a polymorphic variant in the UGT1A1 gene, approved by the FDA (Food and Drug Administration) and included in the recommendations of the European Society of Clinical Oncology (ESMO) for predicting the toxicity of chemotherapy in the treatment of a number of malignant neoplasms, is one of the priorities in pharmacogenetic testing.

The UGT1A1 gene encodes the enzyme uridine diphosphate glucuronosyl transferase I and is responsible for the development of Gilbert syndrome (OMIM 143500). Timely diagnosis of this condition, based on determining the number of tandem repeats in the promoter region of the UGT1A1 gene, is necessary for the prevention of transient hyperbilirubinemia and complications associated with taking a number of drugs.

A widespread method for determining the human genotype by polymorphism UGT1A1 * 6 / * 28 in the promoter region of the UGT1A1 gene, based on the use of specific primers with subsequent detection of PCR results by polyacrylamide gel electrophoresis (http://www.tapotili.ru/topotili-kits .html).

Disadvantages: lack of sensitivity, laboriousness, the need for confirmation by Sanger sequencing.

Another known method is to determine the primary sequence of the promoter region of the UGT1A1 gene by Sanger sequencing (UGT1A1 sequence variants associated with risk of adult hyperbilirubinemia: A quantitative analysis / Chen Zh., Su D., Ai L. Et al. // Gene. - V 552. - 2014 .-- P. 32-38).

Disadvantages: the complexity, the high cost of the study.

There is a method of genotyping variants of the * 28 and * 6 alleles of the human UGT1A1 gene using the pyrosequencing method (UGT1A1 Pyro®, Qiagen, Germany).

Disadvantages: the complexity, the high cost of the study, the need for compatible equipment.

A known method of UGT1A1 testing based on PCR using allele-specific probes (UGT1A1 Genotyping Kit, EntroGen, USA).

Disadvantages: the high cost of the study, the need for compatible equipment.

A known method of determining individual sensitivity to irinotecan using genotyping of allelic variants of the UGT1A1 gene by hybridization on a biological microchip (INFINITI UGT1A1, Autogenomics Inc, USA).

Disadvantages: the complexity, the high cost of the study, the need for compatible equipment.

A known method for determining polymorphisms in the promoter region of the UGT1A1 gene in Gilbert syndrome, based on real-time PCR and detection of the results using the method of analysis of melting curves (HRM) (Minucci, A. Rapid UGT1A1 (TA) (n) genotyping by high resolution melting curve analysis for Gilbert's syndrome diagnosis / Minucci A., Concolino P., Giardina B. et al. // Clin. Chim. Acta. - 2010 .-- V. 411. - P. 246-249). This method is taken by us as a prototype.

Disadvantages: the possibility of use only in the European population.

The objective of the invention is to develop an optimal method for determining the human genotype by polymorphism UGT1A1 * 6 / * 28 in the promoter region of the gene UGT1A1, responsible for the development of toxic side effects in the appointment of a number of chemotherapeutic drugs, as well as associated with the development of Gilbert syndrome, which allows you to diagnose polymorphism UGT1A1 * 6 / * 28 in the human genotype, regardless of national (population) affiliation.

The problem is solved by selecting specific original oligonucleotide sequences for a fragment of the promoter region of the UGT1A1 gene with the desired polymorphic variant and optimizing PCR using an adapted original Taq polymerase buffer.

The technical result of the claimed invention is that it allows you to diagnose polymorphism UGT1A1 * 6 / * 28 in the human genotype, regardless of national (population) affiliation, as well as wide accessibility and high sensitivity at a low research cost. The reaction mixtures include specific original sequences of oligonucleotides, as well as an adapted buffer for Taq polymerase, which increases the sensitivity of the method. The invention allows to reduce the cost of molecular genetic research, to increase its accessibility and testing accuracy through the use of original oligonucleotide sequences and optimized reaction mixtures.

The technical result is achieved by selecting specific original oligonucleotide sequences, optimizing the PCR conditions using the original Taq polymerase buffer composition, and working out the technical conditions for HRM analysis.

The sequence of specific original oligonucleotides is given in the List of sequences of oligonucleotides.

A copy of the list of sequences of oligonucleotides presented on a computer-readable medium is identical to the list of sequences of oligonucleotides in printed form.

The sequences of specific original oligonucleotides are used:

SEQ ID NO 1; SEQ ID NO 2.

Selection of oligonucleotide sequences includes: design of olinucleotides that are strictly complementary to the target DNA sequence, in which there are no polymorphic variants with a minor allele frequency above 0.01% (MAF <0.01).

The composition of the Taq polymerase buffer: Tris-HCl 67 mM, (NH ₄ ) ₂ SO ₄ 166 mM, tween-20 0.1%, glycerol 1%, pH 8.7;

- PCR mode:

Stage 1: denaturation at t = 95 ⁰ C for 5 min

Stage 2: 50 cycles of PCR

- denaturation at t = 95 ⁰ С for 15 sec

- annealing of oligonucleotides: at t = 58 ⁰ C for 15 sec

- elongation at t = 72 ⁰ C for 40 sec

Stage 3: elongation at t = 72 ⁰ for 2 min.

Stage 4: melting of PCR products at t = 72 ⁰ - 95 ⁰ C

Stage 5: cooling and storage at t = 95 ⁰ - 12 ⁰ C.

Detection of a fluorescent signal is carried out both during annealing of olinucleotides on each PCR cycle, and during melting of PCR products with a frequency of 45 to 10 ^° C.

The invention is illustrated by a figure, which shows a graphical image of DNA samples with different genotypes for the allelic variant of UGT1A1 * 28 / * 6.

The invention is illustrated by examples 1 to 3.

Example 1

Name, Patient T.

Age: 1984 (33 g.)

Diagnosis: Cr. ascending colon.

Performed a molecular genetic study according to the claimed method is the determination of the structure of the promoter region of the UGT1A1 gene (2q37), encoding the enzyme uridine diphosphate glucuronosyl transferase I, associated with the development of Gilbert syndrome (OMIM 143500) and sensitivity to irinotecan.

Result: in the analysis of DNA isolated from peripheral blood lymphocytes, the dinucleotide tandem repeat in the promoter part of the UGT1A1 gene is represented by the homozygous genotype UGT1A1 * 6 / * 6: 6TA / 6TA (c.61-6799_61-6798TA NM_205862.1; rs8175347).

Conclusion: This genotype is the norm and is not associated with toxicity with the use of drugs such as irinotecan, rifampicin, tolbutamide and paracetamol.

Example 2

Name, Patient P.

Age: 1950 R. (68 l.)

Diagnosis: Cr. right lung.

Performed a molecular genetic study according to the claimed method is the determination of the structure of the promoter region of the UGT1A1 gene (2q37), encoding the enzyme uridine diphosphate glucuronosyl transferase I, associated with the development of Gilbert syndrome (OMIM 143500) and sensitivity to irinotecan.

Result: in the analysis of DNA isolated from peripheral blood lymphocytes, the dinucleotide tandem repeat in the promoter part of the UGT1A1 gene is represented by the heterozygous genotype UGT1A1 * 6 / * 28: 6TA / 7TA (c.61-6799_61-6798TA NM_205862.1; rs8175347).

Conclusion: Heterozygous polymorphism * 28 6TA / 7TA in the uridine diphosphate-glucuronosyltransferase gene suggests an increased incidence of side effects (hematotoxicity and hepatotoxicity) when irinotecan is used in mono- and combined with PCT platinum preparations. From the use of medium doses, the severity of hematological toxic effects is insignificant, from high doses - pronounced. There is evidence that with a heterozygous genotype to a lesser extent than with a homozygous one, individuals may experience metabolic disorders in drugs such as paracetamol, rifampicin and tolbutamide.

Recommended:

1) dynamic observation:

- biochemical analysis of blood (determination of total and indirect bilirubin in plasma);

- determination of enzyme activity (ALT, AST), alkaline phosphatase;

- determination of the albumin fraction;

2) for the prevention of undesirable drug reactions - an individual selection of the dose of drugs.

Example 3

Name, Patient T.

Age: 1987 year of birth (31 g.)

Diagnosis: Primary multiple malignant neoplasms: Cr. the descending colon. Condition after surgical treatment in 2015 Cr. rectum, mts in the ovary. The condition is in the process of combined treatment.

Performed a molecular genetic study according to the claimed method is the determination of the structure of the promoter region of the UGT1A1 gene (2q37), encoding the enzyme uridine diphosphate glucuronosyl transferase I, associated with the development of Gilbert syndrome (OMIM 143500) and sensitivity to irinotecan.

Result: in the analysis of DNA isolated from peripheral blood lymphocytes, the dinucleotide tandem repeat in the promoter part of the UGT1A1 gene is represented by the homozygous genotype UGT1A1 * 28 / * 28: 7TA / 7TA (c.61-6799_61-6798TA NM_205862.1; rs8175347).

Conclusion: The UGT1A1 * 28 7TA / 7TA genotype is associated with the presence of Gilbert's syndrome (OMIM 143500) in the patient - hereditary non-hemolytic non-conjugated benign hyperbilirubinemia with an autosomal recessive type of inheritance, characterized by an increase in total bilirubin in blood plasma due to indirect fraction up to 20-50 in some cases up to 100) micromol / l against the background of stressful situations, infectious diseases and physical activity.

Homozygous polymorphism * 28 7TA / 7TA in the uridine diphosphate-gluconosyltransferase gene suggests an increased incidence of side effects (hematotoxicity and hepatotoxicity) when irinotecan is used in mono mode and in combination with platinum preparations. In patients carrying a homozygous genotype, metabolic disorders of drugs such as paracetamol, rifampicin and tolbutamide may be observed.

Recommended:

1) dynamic observation:

- biochemical analysis of blood (determination of total and indirect bilirubin in plasma);

- determination of enzyme activity (ALT, AST), alkaline phosphatase;

- determination of the albumin fraction;

2) for the prevention of NLR - an individual selection of the dose of drugs.

List of Oligonucleotide Sequences

<110> FSBI “N.N. Blokhin National Medical Research Center of Oncology ”of the Ministry of Health of the Russian Federation.

<120> A method for determining the human genotype by UGT1A1 * 6 / * 28 polymorphism in the promoter region of the UGT1A1 gene.

<160> NUMBER OF SEQ ID NOS2

<210> SEQ ID NO 1

<211> LENGTH: 23

<212> TYPE: PCR-primer

<213> ORGANISM: Human

<400> SEQUENCE: 1

GAC ACA GTC AAA CAT TAA CTT GG 23

<210> SEQ ID NO: 2

<211> LENGTH: 19

<212> TYPE: PCR-primer

<213> ORGANISM: Human

<400> SEQUENCE: 2

AGA GGT TCG CCC TCT CCT A 19

Claims (1)

A method for determining the human genotype by UGT1A1 * 6 / * 28 polymorphism in the promoter region of the UGT1A1 gene, including real-time PCR with detection of the results using the melting curve analysis (HRM) method, characterized by using specific sequences of oligonucleotides SEQ ID NO: 1 and SEQ ID NO: 2 and PCR optimization using an adapted Taq polymerase buffer composition: Tris-HCl 67 mM, (NH ₄ ) ₂ SO ₄ 166 mM, tween-20 0.1%, glycerol 1%, pH 8.7 .

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